Detection Of Antimicrobial Resistance Genes Research Articles

A new mutation in mgrb mediating polymyxin resistance in Klebsiella variicola

Polymyxin resistance is a public health concern – present in humans, animals and the environment – caused by chromosomal-encoding or plasmid-encoding mechanisms. Chromosomal alterations in MgrB are frequently detected in Klebsiella spp., but not yet reported and characterised in Klebsiella variicola (K. variicola). This study performed microbiological and genomic characterisation of three polymyxin-resistant K. variicola isolates (M14, M15 and M50) recovered from the microbiota of migratory birds in Brazil.The isolates were submitted to SpeI-PFGE, broth microdilution and whole genome sequencing using Illumina MiSeq for analysis of genetic relatedness, sequence typing and detection of antimicrobial-resistance genes. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance only for polymyxins. Sequences of chromosomal two-component systems (PmrAB, PhoPQ, RstAB, CrrAB) and MgrB were evaluated by blastN and blastP against a polymyxin-susceptible K. variicola (A58243), and mutations with biological effect were checked by the PROVEAN tool. K. variicola isolates belonged to two clones, and susceptibility tests showed resistance for polymyxins. In M14 and M15, phoQ deleterious mutations (D90N, I122S and G385S) were identified, while an mgrB variant containing a single deletion (C deletion on position 93) leading to the production of a non-functional protein was detected in M50. mgrB complementation studies showed restoration of polymyxin susceptibility (64 to ≤ 0.25 mg/L) as a wild-type mgrB was inserted into the mgrB-deficient M50.This study confirmed the role of a non-functional mgrB variant in conferring polymyxin resistance, stressing the role of this regulator in K. variicola and drawing attention to novel polymyxin resistance mechanisms emerging in wildlife.

Antibiotic Resistance and Antibiotic Resistance Genes Among Edwardsiella Tarda Isolated from Fish

Edwardsiella tarda is a common fish pathogen, causing significant septicemic diseases in fish. This study was carried out to investigate prevalence of E. tarda among Tilapia zilli at EL manzla lake, Dakahlia governorate and characterize the isolates phenotypically and genotypically in addition to detection of multi-drug resistance genes (B-lactames genes and plasmid-mediated quinolone resistant genes by PCR assay. Therefore, (100) samples of Tilapia zilli collected from different localities. Fish samples were subjected to clinical and post-mortem examination then bacteriological examination from liver, kidney and spleen. The suspected isolates were characterized by cultural and morphological characters, some conventional biochemical tests and API 20E system then by PCR assay. Eighteen % isolates were characterized as E. tarda. Furthermore, detection of antimicrobial resistance genes by PCR, the recovered isolates harbored blaSHV and blaOXA-1 with a prevalence of 50% and 25%, respectively with no detection of aac (6′)-Ib-cr and qepA genes in examined isolates. The results of antibiotics sensitivity showed resistance to Nalidixic acid, Lincomycin, Amoxicillin and Norfloxacin and most of isolate were found to be sensitive to Florfenicol and Neomycin.55.6% of recovered isolates showed resistance to three antibiotic classes and 27.7% of recovered isolates showed resistance to four antibiotic classes and considered as MDR. The emergence of MDR-strains represents a threat-alarm and PCR is very rapid method for identification of E. tarda isolates which may be helpful in control of Edwardsiellosis.

Genomic epidemiology of methicillin-resistant and -susceptible Staphylococcus aureus from bloodstream infections

BackgroundBloodstream infections due to Staphylococcus aureus cause significant patient morbidity and mortality worldwide. Of major concern is the emergence and spread of methicillin-resistant S. aureus (MRSA) in bloodstream infections, which are associated with therapeutic failure and increased mortality.MethodsWe generated high quality draft genomes from 323 S. aureus blood culture isolates from patients diagnosed with bloodstream infection at the Dartmouth-Hitchcock Medical Center, New Hampshire, USA in 2010–2018.ResultsIn silico detection of antimicrobial resistance genes revealed that 133/323 isolates (41.18%) carry horizontally acquired genes conferring resistance to at least three antimicrobial classes, with resistance determinants for aminoglycosides, beta-lactams and macrolides being the most prevalent. The most common resistance genes were blaZ and mecA, which were found in 262/323 (81.11%) and 104/323 (32.20%) isolates, respectively. Majority of the MRSA (102/105 isolates or 97.14%) identified using in vitro screening were related to two clonal complexes (CC) 5 and 8. The two CCs emerged in the New Hampshire population at separate times. We estimated that the time to the most recent common ancestor of CC5 was 1973 (95% highest posterior density (HPD) intervals: 1966–1979) and 1946 for CC8 (95% HPD intervals: 1924–1959). The effective population size of CC8 increased until the late 1960s when it started to level off until late 2000s. The levelling off of CC8 in 1968 coincided with the acquisition of SCCmec Type IV in majority of the strains. The plateau in CC8 also coincided with the acceleration in the population growth of CC5 carrying SCCmec Type II in the early 1970s, which eventually leveled off in the early 1990s. Lastly, we found evidence for frequent recombination in the two clones during their recent clonal expansion, which has likely contributed to their success in the population.ConclusionsWe conclude that the S. aureus population was shaped mainly by the clonal expansion, recombination and co-dominance of two major MRSA clones in the last five decades in New Hampshire, USA. These results have important implications on the development of effective and robust strategies for intervention, control and treatment of life-threatening bloodstream infections.

Antimicrobial Resistance Profiles of Coagulase-Negative Staphylococci in Community-Based Healthy Individuals in Germany.

Coagulase-negative staphylococci (CoNS) are common opportunistic pathogens, but also ubiquitous human and animal commensals. Infection-associated CoNS from healthcare environments are typically characterized by pronounced antimicrobial resistance (AMR) including both methicillin- and multidrug-resistant isolates. Less is known about AMR patterns of CoNS colonizing the general population. Here we report on AMR in commensal CoNS recovered from 117 non-hospitalized volunteers in a region of Germany with a high livestock density. Among the 69 individuals colonized with CoNS, 29 had reported contacts to either companion or farm animals. CoNS were selectively cultivated from nasal swabs, followed by species definition by 16S rDNA sequencing and routine antibiotic susceptibility testing. Isolates displaying phenotypic AMR were further tested by PCR for presence of selected AMR genes. A total of 127 CoNS were isolated and Staphylococcus epidermidis (75%) was the most common CoNS species identified. Nine isolates (7%) were methicillin-resistant (MR) and carried the mecA gene, with seven individuals (10%) being colonized with at least one MR-CoNS isolate. While resistance against gentamicin, phenicols and spectinomycin was rare, high resistance rates were found against tetracycline (39%), erythromycin (33%) and fusidic acid (24%). In the majority of isolates, phenotypic resistance could be associated with corresponding AMR gene detection. Multidrug-resistance (MDR) was observed in 23% (29/127) of the isolates, with 33% (23/69) of the individuals being colonized with MDR-CoNS. The combined data suggest that MR- and MDR-CoNS are present in the community, with previous animal contact not significantly influencing the risk of becoming colonized with such isolates.

Molecular Detection of Carbapenemases in Enterobacterales: A Comparison of Real-Time Multiplex PCR and Whole-Genome Sequencing

Carbapenem-resistant Enterobacterales are a growing problem in healthcare systems worldwide. While whole-genome sequencing (WGS) has become a powerful tool for analyzing transmission and possible outbreaks, it remains laborious, and the limitations in diagnostic workflows are not well studied. The aim of this study was to compare the performance of WGS and real-time multiplex PCR (RT-qPCR) for diagnosing carbapenem-resistant Enterobacterales. In this study, we analyzed 92 phenotypically carbapenem-resistant Enterobacterales, sent to the University Hospital Heidelberg in 2019, by the carbapenem inactivation method (CIM) and compared WGS and RT-qPCR as genotypic carbapenemase detection methods. In total, 80.4% of the collected isolates were identified as carbapenemase producers. For six isolates, discordant results were recorded for WGS, PCR and CIM, as the carbapenemase genes were initially not detected by WGS. A reanalysis using raw reads, rather than assembly, highlighted a coverage issue with failure to detect carbapenemases located in contigs with a coverage lower than 10×, which were then discarded. Our study shows that multiplex RT-qPCR and CIM can be a simple alternative to WGS for basic surveillance of carbapenemase-producing Enterobacterales. Using WGS in clinical workflow has some limitations, especially regarding coverage and sensitivity. We demonstrate that antimicrobial resistance gene detection should be performed on the raw reads or non-curated draft genome to increase sensitivity.

AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence

Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

Isolation, Antimicrobial Resistance Phenotypes, and Virulence Genes of Bordetella bronchiseptica From Pigs in China, 2018-2020.

Bordetella bronchiseptica is a leading cause of respiratory diseases in pigs. However, epidemiological data of B. bronchiseptica in pigs particularly in China, the largest pig rearing country in the world is still limited. We isolated 181 B. bronchiseptica strains from 4259 lung samples of dead pigs with respiratory diseases in 14 provinces in China from 2018 to 2020. The average isolation rate of this 3-year period was 4.25% (181/4259). Antimicrobial susceptibility testing performed by disc diffusion method revealed that most of the B. bronchiseptica isolates in this study were resistant to ampicillin (83.98%), while a proportion of isolates were resistant to cefotaxime (30.39%%), chloramphenicol (12.71%), gentamicin (11.60%), florfenicol (11.60%), tetracycline (8.84%), amoxicillin (8.29%), tobramycin (6.63%), ceftriaxone (4.97%), and cefepime (0.55%). There were no isolates with resistant phenotypes to imipenem, meropenem, polymyxin B, ciprofloxacin, enrofloxacin, and amikacin. In addition, ~13.18% of the isolates showed phenotypes of multidrug resistance. Detection of antimicrobial resistance genes (ARGs) by PCR showed that 16.57% of the B. bronchiseptica isolates in this study was positive to aac(3)-IV, while 3.87%, 2.21%, 1.10%, 0.55%, 0.55%, and 0.55% of the isolates were positive to aac6'-Ib, rmtA, blaTEM, blaSHV, oqxB, and tetA, respectively. Detection of virulence factors encoding genes (VFGs) by conventional PCR showed that over 90% of the pig B. bronchiseptica isolates in this study were positive to the five VFGs examined (fhaB, 97.24%; prn, 91.16%; cyaA, 98.34%; dnt, 98.34%; betA, 92.82%). These results demonstrate B. bronchiseptica as an important pathogen associated with pig respiratory disorders in China. The present work contributes to the current understanding of the prevalence, antimicrobial resistance and virulence genes of B. bronchiseptica in pigs.

WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

BackgroundBacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools.ResultsPublicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the “Flye” assembler with “meta” settings generated the most complete plasmids.ConclusionsWeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.

Antimicrobial Resistance Gene Detection Methods for Bacteria in Animal-Based Foods: A Brief Review of Highlights and Advantages.

Antimicrobial resistance is a major public health problem and is mainly due to the indiscriminate use of antimicrobials in human and veterinary medicine. The consumption of animal-based foods can contribute to the transfer of these genes between animal and human bacteria. Resistant and multi-resistant bacteria such as Salmonella spp. and Campylobacter spp. have been detected both in animal-based foods and in production environments such as farms, industries and slaughterhouses. This review aims to compile the techniques for detecting antimicrobial resistance using traditional and molecular methods, highlighting their advantages and disadvantages as well as the effectiveness and confidence of their results.

A shotgun metagenomics approach to detect and characterize unauthorized genetically modified microorganisms in microbial fermentation products

The presence of a genetically modified microorganism (GMM) or its DNA, often harboring antimicrobial resistance (AMR) genes, in microbial fermentation products on the market is prohibited by European regulations. GMMs are currently screened for through qPCR assays targeting AMR genes and vectors, and then confirmed by targeting known specific GM constructs/events. However, when the GMM was not previously characterized and an isolate cannot be obtained, its presence cannot be proven. We present a metagenomics approach capable of delivering the proof of presence of a GMM in a microbial fermentation product, with characterization based on the detection of AMR genes and vectors, species and unnatural associations in the GMM genome. In our proof-of-concept study, this approach was performed on a case with a previously isolated and sequenced GMM, an unresolved case for which no isolate was obtained, and a non-GMM-contaminated sample, all representative for the possible scenarios to occur in routine setting. Both short and long read sequencing were used. This workflow paves the way for a strategy to detect and characterize unknown GMMs by enforcement laboratories.

Outcome of Different Sequencing and Assembly Approaches on the Detection of Plasmids and Localization of Antimicrobial Resistance Genes in Commensal Escherichia coli.

Antimicrobial resistance (AMR) is a major threat to public health worldwide. Currently, AMR typing changes from phenotypic testing to whole-genome sequence (WGS)-based detection of resistance determinants for a better understanding of the isolate diversity and elements involved in gene transmission (e.g., plasmids, bacteriophages, transposons). However, the use of WGS data in monitoring purposes requires suitable techniques, standardized parameters and approved guidelines for reliable AMR gene detection and prediction of their association with mobile genetic elements (plasmids). In this study, different sequencing and assembly strategies were tested for their suitability in AMR monitoring in Escherichia coli in the routines of the German National Reference Laboratory for Antimicrobial Resistances. To assess the outcomes of the different approaches, results from in silico predictions were compared with conventional phenotypic- and genotypic-typing data. With the focus on (fluoro)quinolone-resistant E. coli, five qnrS-positive isolates with multiple extrachromosomal elements were subjected to WGS with NextSeq (Illumina), PacBio (Pacific BioSciences) and ONT (Oxford Nanopore) for in depth characterization of the qnrS1-carrying plasmids. Raw reads from short- and long-read sequencing were assembled individually by Unicycler or Flye or a combination of both (hybrid assembly). The generated contigs were subjected to bioinformatics analysis. Based on the generated data, assembly of long-read sequences are error prone and can yield in a loss of small plasmid genomes. In contrast, short-read sequencing was shown to be insufficient for the prediction of a linkage of AMR genes (e.g., qnrS1) to specific plasmid sequences. Furthermore, short-read sequencing failed to detect certain duplications and was unsuitable for genome finishing. Overall, the hybrid assembly led to the most comprehensive typing results, especially in predicting associations of AMR genes and mobile genetic elements. Thus, the use of different sequencing technologies and hybrid assemblies currently represents the best approach for reliable AMR typing and risk assessment.

A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

Whole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

Molecular Detection of Antimicrobial Resistance Genes and Virulence Genes in E. coli Isolated from Sheep and Goat Faecal Samples

Background: E. coli is a Gram negative bacterium commonly found in the intestine of animals and humans. Most E. coli are harmless but some can cause infection. Antimicrobial resistance is a public health problem, hence this study was carried out to estimate the antimicrobial resistance pattern and virulotyping of E. coli isolates.Methods: During the period 2018-19, a total of 100 faecal samples from goats (n=50) and sheep (n=50) were processed for isolation and identification of E.coli. All the E. coli isolates were tested for resistance against 10 different antibiotics. The prevalence of various antibiotic resistance genes viz bla TEM, tetA and tetB and virulence genes Stx1, Stx2 and hlyA in all E. coli isolates was determined by PCR.Result: Out of 100 samples, 40 isolates of E.coli (24 from goats and 16 from sheep) were isolated, identified biochemically and by PCR. Majority of the E.coli isolates were found resistant to penicillin (100%) followed by ampicillin (93%), tetracycline, (68.8%), gentamicin (37.4%), ceftriazone (18.8%), amikacin (16.7%), chloramphenicol (4.2%), co- trimoxazole (4.2%) and ofloxacin (4.2%). Out of all the isolates, 12 isolates were positive for blaTEM, 10 for tetB, 19 for tetA, five for Stx1 and hlyA and none carried Stx2 gene.

313. Using a Clinical Decision Prediction Tool to Improve Empirical Antimicrobial Therapy in Ceftriaxone-Resistant Enterobacterales Bloodstream Infections

BackgroundThere are several clinical tools for prediction of antimicrobial resistance. However, their utility in management of bloodstream infections (BSI) due to resistant bacteria remains unclear. This quasi-experimental cohort examined the impact of utilization of the extended-spectrum beta-lactamase (ESBL) prediction score on time to initiation of appropriate antimicrobial therapy (AAT) in BSI due to ceftriaxone-resistant (CRO-R) Enterobacterales.MethodsAdults with first episodes of monomicrobial BSI due to CRO-R Enterobacterales at Prisma Health-Midlands Hospitals in South Carolina from January 2010 to December 2017 were included. Antimicrobial stewardship intervention was implemented in January 2014 consisting of real-time alerts for positive blood cultures and rapid diagnostics for identification of bloodstream isolates. The ESBL prediction score was used to stratify risk of resistance prior to conventional antimicrobial susceptibility results. Student’s t-test was used to compare mean time to AAT before and after intervention.ResultsAmong 92 patients with BSI due to CRO-R Enterobacterales, median age was 66 years, 52 (57%) were men, 68 (74%) had community-onset BSI, and 52 (57%) had urinary source of infection. Escherichia coli 52 (57%) and Klebsiella species 27 (29%) were the most common bloodstream isolates and the majority 63 (68%) produced ESBLs. There were no significant differences in demographics or clinical characteristics of patients before (n=45) and after (n=47) antimicrobial stewardship intervention. Mean time to AAT was 3.2 days prior and 1.7 days after utilization of ESBL prediction score (p=0.021). Results were consistent among ESBL-producing Enterobacterales (time to AAT 2.8 before and 1.8 days after intervention, p=0.025).ConclusionUtilization of the ESBL prediction score as part of a real-time antimicrobial stewardship intervention significantly reduced time to AAT in BSI due to CRO-R and ESBL-producing Enterobacterales. The ESBL prediction score represents a viable tool to improve antimicrobial management in clinical settings that lack access to multiplex PCRs for detection of antimicrobial resistance genes and novel methods for rapid phenotypic antimicrobial susceptibility testing.DisclosuresJulie Ann Justo, PharmD, MS, BCPS-AQ ID, bioMerieux (Speaker’s Bureau)TRC Healthcare (Speaker’s Bureau)

640. Analytical Validation of the BioFire® Bone and Joint Infection (BJI) Panel for the Identification of Bacteria, Yeast, and Antimicrobial Resistance Genes from Synovial Fluid

BackgroundThe BioFire Bone and Joint Infection (BJI) Panel is a sample-to-answer test for the qualitative detection of nearly 40 different bacteria, yeast, and antimicrobial resistance (AMR) genes in synovial fluid (SF). The panel aims to improve on current culture-based diagnostics, particularly for detection of anaerobes (e.g. Finegoldia magna, Kingella kingae, Cutibacterium, Anaerococcus and Peptoniphilus species, and others) in about an hour. Analytical performance of the panel (Limit of Detection (LoD), analytical reactivity and specificity, interference, reproducibility), and specimen storage conditions are described.MethodsLoD for each analyte was estimated from serial dilutions and confirmed at the lowest titer with ≥95% detection. A collection of >350 isolates representing genetic and geographic diversity of analytes was tested near LoD to assess analytical reactivity, and more than 420 near-neighbor, commensal, pathogenic, or environmental off-panel species were evaluated for assay specificity. Reproducibility was evaluated in a multi-laboratory multi-variable study, and the impact of storage and potentially interfering substances on the accuracy of test results was also assessed. Testing was performed with Investigational Use Only kits.ResultsThe confirmed LoD for bacteria and yeast ranged from 100 - 10,000 CFU/mL. Sequence analysis and testing demonstrated clinically appropriate specificity and reactivity with a variety of isolates and different AMR gene types. Accurate and reproducible organism and AMR gene detection was observed with repeated testing of samples over several days (99.9% agreement with the expected results), and detection was not affected by potentially interfering substances nor by refrigerated sample storage.ConclusionThe BioFire BJI Panel is a robust, accurate, and easy-to-use multiplex PCR test capable of detecting many aerobic and anaerobic bacteria, yeast, and AMR genes in synovial fluid specimens. Rapid and reliable molecular detection of possible BJI pathogens may advance the diagnosis and effective management of bone and joint infections.NoteThis panel has not been evaluated by the FDA or other regulatory agencies for diagnostic use.DisclosuresNicholas Francis, n/a, BioFire Diagnostics (Employee) Laurence Barbier, n/a, Biomerieux (Employee) Caroline Dubost, n/a, Biomerieux (Employee) Elodie Billet, n/a, Biomerieux (Employee) Joel Manwaring, n/a, BioFire Diagnostics (Employee) Josh Southwick, n/a, BioFire Diagnostics (Employee) Tyson Dawson, n/a, BioFire Diagnostics (Employee) Jess Gann, n/a, BioFire Diagnostics (Employee) Kevin Ekins, n/a, BioFire Diagnostics (Employee) Jennifer Arce, MS, BioFire Diagnostics/BioMerieux (Employee) Briana Flaherty, n/a, BioFire Diagnostics (Employee) Harmonie Durand, n/a, Biomerieux (Employee) Chris Cantrell, n/a, Biomerieux (Employee) Elizabeth Amiott, n/a, BioFire Diagnostics (Employee)

1078. Renal Transplant Recipient Resistomes Reveal Expansive Sub-Clinical Burden of Resistance After Treatment for ESBL-Producing Bacterial Infections

BackgroundRenal transplant recipients have frequent infection and colonization with antibiotic resistant (AR) bacteria. However, little is known about the burden of AR following targeted antibiotic treatment.MethodsThis was a prospective study conducted as part of a single center clinical trial at Emory University. Demographic and clinical data regarding transplant and AR bacterial infection were abstracted. Stool samples were collected from renal transplant recipients treated with antibiotics for ESBL-producing gram negative infections. Bacterial cultures with AR-selective media and Illumina short-read sequencing were performed on stool samples. Confirmatory phenotypic isolate AR testing was performed with the Vitek2 platform. Resistome profiles were produced by assembling short reads into scaffolds using MetaSPAdes, predicting protein coding sequences using Prodigal and classifying proteins as antimicrobial resistance determinants using AMRFinderPlus. AMRFinderPlus results for patients were then compared to fecal metagenomes from 3 healthy Human Microbiome Project controls. Differences in AR genes in renal transplant patients vs controls were compared.ResultsMetagenome sequencing was performed for 6 (5 female) patient stool samples. Stools were collected a median of 30 days after infection. The median number of AR genes per patient metagenome was 48.5 (range 23 to 87 genes). The median number of AR genes per control metagenome was 24 (range 16 to 25 genes). We detected 97 unique AR genes across all samples, 63 of which (65%) were detected in patient samples but not controls. All AR genes found in control metagenomes were present in at least one patient metagenome. No AR genes detected in patients were common to all patients. Subsets of clinically relevant genes corresponded with patient stool AR bacteria culture results.Antimicrobial resistance gene detection heatmap for renal transplant recipient stool samples after antibiotic treatment for ESBL infection. ConclusionViable AR bacteria and diverse AR gene profiles were frequently detected from renal transplant recipient stool samples after antibiotic treatment for infection. These data suggest that AR bacterial colonization and AR gene profiles may require distinct treatments other than systemic antibiotics for eradication.Disclosures All Authors: No reported disclosures

Evaluation of Microbiological Performance and the Potential Clinical Impact of the ePlex® Blood Culture Identification Panels for the Rapid Diagnosis of Bacteremia and Fungemia.

Molecular rapid diagnostic assays associated with antimicrobial stewardship have proven effective for the early adaptation of empiric therapy in bloodstream infections. The ePlex® BCID (GenMark Diagnostics) Panels allow identification of 56 bacteria and fungi and 10 resistance genes in 90 min directly from positive blood cultures. We prospectively evaluated 187 sepsis episodes at Grenoble University Hospital and retrospectively analyzed the cases to measure the potential clinical impact of the ePlex BCID results. Identification of all pathogens was obtained for 164/187 (88%) bloodstream infections with 100% detection of antimicrobial resistance genes (17 blaCTX-M, 1 vanA, and 17 mecA genes). Only 15/209 (7%) strains were not covered by the panels. Sensitivity for detection of micro-organisms targeted by the RUO BCID-GP, BCID-GN, and BCID-FP Panels was respectively 84/84 (100%), 103/107 (96%), and 14/14 (100%). Interestingly, accurate identification of all pathogens was achieved in 15/17 (88%) polymicrobial samples. Retrospective analysis of medical records showed that a modification of antimicrobial treatment would have been done in 45% of the patients. Treatment modifications would have been an optimization of empiric therapy, a de-escalation or an escalation in respectively 16, 17, and 11% of the patients. Moreover, 11% of the samples were classified as contaminants or not clinically relevant and would have led to early de-escalation or withdrawal of any antibiotic. Detection of resistance genes in addition to identification alone increased escalation rate from 4 to 11% of the patients. Absence of the ePlex result was considered a lost opportunity for therapy modification in 28% of patients.

Antimicrobial Resistance Gene Detection and Plasmid Typing Among Multidrug Resistant Enterococci Isolated from Freshwater Environment.

In this study, mechanisms of antimicrobial resistance (AR) as well as the abundance and diversity of plasmids were determined among multidrug resistant (MDR) enterococci from surface water in GA, USA. A total of 51 enterococci isolates were screened for the presence of 27 AR genes conferring resistance to ciprofloxacin, erythromycin, tylosin, kanamycin, streptomycin, lincomycin, Quinupristin/Dalfopristin (Q/D), and tetracycline. A plasmid classification system based on replication genes was used to detect 19 defined Gram-positive plasmid replicon families. Twelve genes were identified as conferring resistance to erythromycin and tylosin (erm(B) and erm(C)), kanamycin (aph(3′)-IIIa), streptomycin (ant(6)-Ia), lincomycin (lnu(B)), Q/D (vat(E)), ciprofloxacin (qnrE. faecalis), and tetracycline (tet(K), tet(L), tet(M), tet(O) and tet(S)). Twelve different rep-families were identified in two-thirds of the isolates. While AR genes commonly found in human and animals were detected in this study among environmental enterococci, resistance genes could not be determined for many of the isolates, which indicates that diverse AR mechanisms exist among enterococci, and the understanding of AR mechanisms for environmental enterococci is limited. Diverse rep-families were identified among the enterococci recovered from the aquatic environment, and these rep-families appear to be quite different from those recovered from other sources. This work expands knowledge of AR gene reservoirs and enterococcal plasmids across a wider range of environments.

Genetic Identification and Drug-Resistance Characterization of Mycobacterium tuberculosis Using a Portable Sequencing Device. A Pilot Study.

Clinical management of tuberculosis (TB) in endemic areas is often challenged by a lack of resources including laboratories for Mycobacterium tuberculosis (Mtb) culture. Traditional phenotypic drug susceptibility testing for Mtb is costly and time consuming, while PCR-based methods are limited to selected target loci. We herein utilized a portable, USB-powered, long-read sequencing instrument (MinION), to investigate Mtb genomic DNA from clinical isolates to determine the presence of anti-TB drug-resistance conferring mutations. Data analysis platform EPI2ME and antibiotic-resistance analysis using the real time ARMA workflow, identified Mtb species as well as extensive resistance gene profiles. The approach was highly sensitive, being able to detect almost all described drug resistance conferring mutations based on previous whole genome sequencing analysis. Our findings are supportive of the practical use of this system as a suitable method for the detection of antimicrobial resistance genes, and effective in providing Mtb genomic information. Future improvements in the error rate through statistical analysis, drug resistance prediction algorithms and reference databases would make this a platform suited for the clinical setting. The small size, relatively inexpensive cost of the device, as well as its rapid and simple library preparation protocol and analysis, make it an attractive option for settings with limited laboratory infrastructure.

Detection of Antimicrobial Resistance Genes in the Milk Production Environment: Impact of Host DNA and Sequencing Depth.

Over the past decades, antimicrobial resistance (AMR) has been recognized as one of the most serious threats to public health. Although originally considered a problem to human health, the emerging crisis of AMR requires a “One Health” approach, considering human, animal, and environmental reservoirs. In this regard, the extensive use of antibiotics in the livestock production systems to treat mastitis and other bacterial diseases can lead to the presence of AMR genes in bacteria that contaminate or naturally occur in milk and dairy products, thereby introducing them into the food chain. The recent development of high-throughput next-generation sequencing (NGS) technologies is improving the fast characterization of microbial communities and their functional capabilities. In this context, whole metagenome sequencing (WMS), also called shotgun metagenomic sequencing, allows the generation of a vast amount of data which can be interrogated to generate the desired evidence, including the resistome. However, the amount of host DNA poses a major challenge to metagenome analysis. Given the current absence of literature concerning the application of WMS on milk to detect the presence of AMR genes, in the present study, we evaluated the effect of different sequencing depths, host DNA depletion methods and matrices to characterize the resistome of a milk production environment. WMS was conducted on three aliquots of bulk tank milk and three aliquots of the in-line milk filter collected from a single dairy farm; a fourth aliquot of milk and milk filter was bioinformatically subsampled. Two commercially available host DNA depletion methods were applied, and metagenomic DNA was sequenced to two different sequencing depth. Milk filters proved to be the most suitable matrices to evaluate the presence of AMR genes; besides, the pre-extraction host DNA depletion method was the most efficient approach to remove host reads. To our knowledge, this is the first study to evaluate the limitations posed by the host DNA in investigating the milk resistome with a WMS approach, confirming the circulation of AMR genes in the milk production environment.